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Dual inhibition of TMPRSS2 and Cathepsin Bprevents SARS-CoV-2 infection in iPS cells.

ABSTRACT: It has been reported that many receptors and proteases are required for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Although angiotensin-converting enzyme 2 (ACE2) is the most important of these receptors, little is known about the contribution of other genes. In this study, we examined the roles of neuropilin-1, basigin, transmembrane serine proteases (TMPRSSs), and cathepsins (CTSs) in SARS-CoV-2 infection using the CRISPR interference system and ACE2-expressing human induced pluripotent stem (iPS) cells. Double knockdown of TMPRSS2 and cathepsin B (CTSB) reduced the viral load to 0.036% ± 0.021%. Consistently, the combination of the CTPB inhibitor CA-074 methyl ester and the TMPRSS2 inhibitor camostat reduced the viral load to 0.0078% ± 0.0057%. This result was confirmed using four SARS-CoV-2 variants (B.1.3, B.1.1.7, B.1.351, and B.1.1.248). The simultaneous use of these two drugs reduced viral load to less than 0.01% in both female and male iPS cells. These findings suggest that compounds targeting TMPRSS2 and CTSB exhibit highly efficient antiviral effects independent of gender and SARS-CoV-2 variant.

SUBMITTER: Hashimoto R

PROVIDER: S-EPMC8527102 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using nonspecific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high-throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

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Dual inhibition of TMPRSS2 and Cathepsin Bprevents SARS-CoV-2 infection in iPS cells.

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